Solid salt forms of a pyrrole substituted 2-indolinone

ABSTRACT

The present invention relates to solid salt forms of the 3-pyrrole substituted 2-indolinone compound 5-[5-fluoro-2-oxo-1, 2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pirrolidin-1-yl-ethyl)-amide. It also relates to polymorphs of the phosphate salt of the amide. The invention further relates to the use of the salts and polymorphs in the treatment of protein kinase related disorders.

This Application is a continuation of U.S. patent application Ser. No.12/067,242, filed Mar. 18,2008, now pending, which was filed under 35USC 371 as a US National Stage Patent Application of InternationalPatent Application No. PCT/IB2006/002506, filed Sep. 8, 2006 under 35USC 363, now abandoned, which claims the benefit under 35 USC 119(e)(l)of US provisional Patent Application No. 60/718,586, filed Sep. 19, 2005under 35 USC 111(b), now abandoned, the entire disclosures of which areincorporated herein by reference.

FIELD OF INVENTION

The present invention relates to solid salt forms of a 3-pyrrolesubstituted 2-indolinone compound,5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide.The foregoing compounds modulate the activity of protein kinases(“PKs”). The compounds of this invention are therefore useful intreating disorders related to abnormal PK activity. Pharmaceuticalcompositions comprising salts of this compound and methods of preparingthem are disclosed. The present invention is also directed to polymorphsof the phosphate salt form of the amide.

BACKGROUND

The following is offered as background information only and is notadmitted to be prior art to the present invention.

Solids, including pharmaceuticals, often have more than one crystalform, and this is known as polymorphism. Polymorphism occurs when acompound crystallizes in a multiplicity of solid phases that differ incrystal packing. Numerous examples are cited in the standard referencesof solid state properties of pharmaceuticals, Byrn, S. R., Solid-StateChemistry of Drugs, New Your, Academic Press (1982);Kuhnert-Brandstatter, M., Thermomiscroscopy In The Analysis ofPharmaceuticals, New York, Pergamon Press (1971) and Haleblian, J. K.and McCrone, W. Pharmaceutical applications of polymorphism. J. Pharm.Sci., 58, 911 (1969). Byrn states that, in general, polymorphs exhibitdifferent physical characteristics including solubility and physical andchemical stability.

Because of differences in molecular packing, polymorphs may differ inways that influence drug release, solid-state stability, andpharmaceutical manufacturing. The relative stability and theinterconversions of polymorphs are particularly important to theselection of a marketed drug. A suitable polymorph may hinge upon theissue of physical stability. For example, the selection of a marketeddrug may depend upon the availability and selection of a suitablepolymorph having desirable characteristics, such as excellent physicalstability or the ability to be manufactured in large scale. Theperformance of the solid dosage form should not be limited bypolymorphic transformations during the shelf life of the product. It isimportant to note that there is no reliable method to predict theobservable crystal structures of a given drug or to predict theexistence of polymorphs with desirable physical properties.

PKs are enzymes that catalyze the phosphorylation of hydroxy groups ontyrosine, serine, and threonine residues of proteins. The consequencesof this seemingly simple activity are staggering since virtually allaspects of cell life (e.g., cell growth, differentiation, andproliferation) in one way or another depend on PK activity. Furthermore,abnormal PK activity has been related to a host of disorders, rangingfrom relatively non-life threatening diseases such as psoriasis toextremely virulent diseases such as glioblastoma (brain cancer).

Receptor tyrosine kinases (RTKs), a class of PK, are excellentcandidates for molecular targeted therapy, because they play key rolesin controlling cell proliferation and survival and are frequentlydysregulated in a variety of malignancies. The mechanisms ofdysregulation include overexpression (Her2/neu in breast cancer,epidermal growth factor receptor in non-small cell lung cancer),activating mutations (KIT in gastrointestinal stromal tumors,fms-related tyrosine kinase 3/Flk2 (FLT3) in acute myelogenousleukemia), and autocrine loops of activation (vascular endothelialgrowth factorNEGF receptor (VEGF/VEGFR) in melanoma, platelet-derivedgrowth factor/PDGF receptor (PDGF/PDGFR) in sarcoma).

Aberrantly regulated RTKs have been described in comparable human andcanine cancers. For example, aberrant expression of the Met oncogeneoccurs in both human and canine osteosarcoma. Interestingly, comparableactivating mutations in the juxtamembrane (JM) domain of c-kit are seenin 50-90% of human gastrointestinal stromal tumors (GISTs) and in 30-50%of advanced canine MCTs (mast cell tumors). Although the mutations inhuman GISTs consist of deletions in the JM domain and those in canineMCTs consist of internal tandem duplications (ITDs) in the JM domain,both lead to constitutive phosphorylation of KIT in the absence ofligand binding. The RTKs and their ligands, VEGF, PDGF, and FGF mediateneo-vascularization, known as angiogenesis, in solid tumors.Consequently, by inhibiting the RTKs, the growth of new blood vesselsinto tumors may be inhibited.

Antiangiogenesis agents, a class of molecules that inhibits the growthof blood vessels into tumors, have much less toxicity to the bodycompared to conventional anti-cancer drugs. U.S. Pat. No. 6,573,293,incorporated herein by reference, discloses, among other compounds,5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)-amide (hereinafter “Compound I”). It hasthe following structure:

Compound I is a small molecule that exhibits PK modulating ability. Thecompound is therefore useful in treating disorders related to abnormalPK activity. It is an inhibitor of the RTKs, PDGFR, VEGFR, KIT, andFLT3. Compound I has been shown to inhibit KIT phosphorylation, arrestcell proliferation, and induce cell cycle arrest and apoptosis inmalignant mast cell lines in vitro expressing various forms of mutantKIT. Compound I and related molecules are effective in preclinicalmodels against tumor xenografts arising from cell lines of diverse humantumor origin.

Compound I is useful for treating cancers in companion animals, mainlydogs, and is also useful for the treatment of, inter alia, cancer inhumans. Such cancers include, but are not limited to, leukemia, braincancer, non-small cell lung cancer, squamous cell carcinoma,astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladdercancer, head and neck cancer, small-cell lung cancer, glioma, colorectalcancer, genitourinary cancer, and gastrointestinal stromal cancer. Also,Compound I is useful for the treatment of diseases related tooverexpression of mast cells, including but not limited to, mastocytosisin humans and mast cell tumors in dogs.

Compound I was recently shown to be clinically effective against anumber of spontaneous malignancies in dogs. In the study, 11 of 22canine MCTs showed durable objective responses (partial responses andcomplete responses) to Compound I treatment; 9 of these MCTs possessedITDs in the JM domain of c-kit.

Compound I readily crystallizes. Its solubility is about 10 μg/mL in pH6 phosphate buffer at 25° C. When the compound was synthesized, veryfine particles precipitated out of solution during the last step ofsynthesis. Subsequent isolation of these fine particles by filtrationwas slow, and a hard cake resulted after filtration. There is a need fora salt of Compound I which has physical stability and desirable physicalproperties.

SUMMARY OF THE INVENTION

This invention comprises salt forms of Compound I. Five different saltforms of Compound I were synthesized and are described herein. (SeeTable 1) These include the hydrochloride, fumarate, citrate, phosphate,and ascorbate salts of Compound I. Based on characterization of thesesalts, the 1:1 phosphate salt, Compound I phosphate, was identified as asalt form with highly desirable characteristics. Polymorph screeningrevealed the existence of 10 polymorphs of Compound I phosphate, hereinnamed Forms I through X.

In one aspect, this invention provides two salt forms of Compound I,wherein the salt form is selected from the citrate and phosphate salts,and solvates and polymorphs thereof. In one embodiment, the phosphatesalt form with a molecular formula of C₂₂H₂₅FN₄O2H₃O₄P is selected. Inanother embodiment, the phosphate salt form with a melting point fromabout 285 to about 290° C. is selected. Compound I phosphate has astructure of

In another embodiment, the citrate salt, Compound I citrate, which has amolecular formula of C₂₂H₂₅FN₄O2C₆H₈O₇ is selected. In yet anotherembodiment, the citrate salt form with a melting point from about 178 toabout 183° C. is selected. Compound I citrate has a structure of

A second aspect of the invention is a pharmaceutical compositioncomprising the phosphate salt or the citrate salt of Compound I, orsolvates or polymorphs thereof, and a pharmaceutically acceptablecarrier or excipient.

A third aspect of the invention is a method for the modulation of thecatalytic activity of protein kinases comprising contacting said proteinkinase with the phosphate or citrate salts of Compound I, or solvates orpolymorphs thereof. The protein kinase may be selected from the groupconsisting of receptor tyrosine kinases, non-receptor protein tyrosinekinases, and serine/threonine protein kinases.

A fourth aspect of the invention is a method of preventing or treating aprotein kinase related disorder in an organism comprising administeringto said organism a therapeutically effective amount of a pharmaceuticalcomposition comprising the phosphate salt or the citrate salt ofCompound I, or solvates or polymorphs thereof, and a pharmaceuticallyacceptable carrier or excipient. In one embodiment, the organism is ahuman. In another embodiment, the organism is a companion animal. Instill another embodiment, the companion animal is a cat or a dog. Theprotein kinase related disorder may be selected from the groupconsisting of a receptor tyrosine kinase related disorder, anon-receptor protein tyrosine kinase related disorder, and aserine/threonine protein kinase related disorder. The protein kinaserelated disorder may be selected from the group consisting of an EGFRrelated disorder, a PDGFR related disorder, an IGFR related disorder, ac-kit related disorder, and a FLK related disorder. Such disordersinclude by way of example and not limitation, leukemia, brain cancer,non-small cell lung cancer, squamous cell carcinoma, astrocytoma,Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, headcancer, neck cancer, melanoma, ovarian cancer, prostate cancer, breastcancer, small cell lung cancer, glioma, mastocytosis, mast cell tumor,colorectal cancer, genitourinary cancer, gastrointestinal cancer,diabetes, an autoimmune disorder, a hyperproliferation disorder,restenosis, fibrosis, psoriasis, von Heppel-Lindau disease,osteoarthritis, rheumatoid arthritis, angiogenesis, an inflammatorydisorder, an immunological disorder, and a cardiovascular disorder.

A fifth aspect of the invention is a method of preparing phosphate saltcrystals of base5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)-amide which comprises introducing astoichiometric amount of phosphoric acid to the base in a solutioncomprising a solvent or a mixture of solvents, forcing the phosphatesalt in solution to crystallize, separating the phosphate salt crystalsfrom the solvent solution, and drying the crystals. The phosphoric acidmay be introduced in an amount which is 40% molar excess to the base.The solvent may comprise isopropanol. The step of separating thecrystals from the solvent solution may comprise adding acetonitrile tothe solution and rotovapping the solution. The step of separating thecrystals from the solvent solution may also comprise filtration.

A sixth aspect of the invention is a method of preparing citrate saltcrystals of base5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)-amide which comprises introducing astoichiometric amount of citric acid to the base in a solutioncomprising a solvent or a mixture of solvents, forcing the citrate saltin solution to crystallize, separating the citrate salt crystals fromthe solvent solution, and drying the crystals. The citric acid may alsobe introduced in an amount of about 40% molar excess to the base. Thesolvent may comprise methanol. The step of separating the crystals fromthe solvent solution may comprise adding acetonitrile to the solutionand rotovapping the solution. The step of separating the crystals fromthe solvent solution may comprise filtration.

In a seventh aspect, the invention provides the polymorphs Forms I-X (asdescribed herein) of the phosphate salt of Compound I. In oneembodiment, Form I is provided.

An eighth aspect of the invention is a pharmaceutical compositioncomprising the Form I polymorph of Compound I phosphate and apharmaceutically acceptable carrier or excipient.

A ninth aspect of the invention is a method for the modulation of thecatalytic activity of protein kinases comprising contacting said proteinkinase with the Form I polymorph of Compound I phosphate.

A tenth aspect of the invention is a method of preventing or treating aprotein kinase related disorder in an organism comprising administeringto said organism a therapeutically effective amount of the Form Ipolymorph of Compound I phosphate. In one embodiment, the organism is ahuman or companion animal. In another embodiment, the companion animalis a cat or a dog. Such disorders include by way of example and notlimitation, mast cell tumor and mastocytosis.

An eleventh aspect of the invention is a method of preparing polymorphsof Compound I phosphate, which comprises introducing the phosphate saltto a solution comprising a solvent or a mixture of solvents, optionally,adding a bridging solvent to the solution, and separating the polymorphcrystals from the solvent solution. The solution may comprise water plusacetonitrile. The solution may comprise methanol. The bridging solventmay be methanol.

A twelfth aspect of the invention is the use of the phosphate or citratesalts of Compound I or the Form I polymorph of the phosphate salt in thepreparation of a medicament which is useful in the treatment of adisease mediated by abnormal PK activity.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Moisture sorption data for salts of Compound I.

FIG. 2. Powder X-ray Diffraction patterns for Compound I citrate andCompound I phosphate.

FIG. 3. Powder X-ray Diffraction patterns of the ten unique solidsobtained from the polymorph screening study (See Example 5). Form Ithrough Form X as designated in Tables 5 and 6 are presented.

FIG. 4. TGA curves of solids from CH₂C1₂ (Form VI, immediatelyafter,precipitation), Hexane (Form VII, after standing overnight), andacetonitrile (Form VIII, after standing 3 days).

FIG. 5. Results of agarose gel electrophoresis of PCR products from MCTsevaluated in Example 7. Lanes 1-5 correspond to patients 1-5 in Table 8;Lanes 6-14 correspond to patients 6-14 in Table 8. Controls consisted ofPCR products generated from the C2 canine mast cell line containing a48-bp ITD (Lane 15) and from normal canine cerebellum (wild type; Lane16).

FIG. 6. Reductions in MCT phosphorylated KIT and phosphorylatedextracellular signal-regulated kinase (ERK) 1/2 after a single dose ofCompound I phosphate

DETAILED DESCRIPTION OF THE INVENTION

Definitions. Unless otherwise stated the following terms used in thespecification and claims have the meanings discussed below:

The term “C” when used in reference to temperature means centigrade orCelsius.

The term “catalytic activity” refers to the rate of phosphorylation oftyrosine under the influence, direct or indirect, of RTKs and/or CTKs orthe phosphorylation of serine and threonine under the influence, director indirect, of STKs.

The term “companion animal” refers to domesticated animals offeringcompanionship to humans, and includes, but is not limited to, cats anddogs.

The term “contacting” refers to bringing a compound of the presentinvention and a target PK together in such a manner that the compoundcan affect the catalytic activity of the PK, either directly, i.e., byinteracting with the kinase itself, or indirectly, i.e., by interactingwith another molecule on which the catalytic activity of the kinase isdependent.

The term “IC₅₀” means the concentration of a test compound whichachieves a half-maximal inhibition of the PK activity.

The term “modulation” or “modulating” refers to the alteration of thecatalytic activity of RTKs, CTKs, and STKs. In particular, modulatingrefers to the activation or inhibition of the catalytic activity ofRTKs, CTKs, and STKs, preferably the activation of the catalyticactivity of RTKs, CTKs, and STKs, depending on the concentration of thecompound or salt to which the RTK, CTK, or STK is exposed or, morepreferably, the inhibition of the catalytic activity of RTKs, CTKs, andSTKs.

The term “PK” refers to receptor protein tyrosine kinase (RTKs),non-receptor or “cellular” tyrosine kinase (CTKs) and serine-threoninekinases (STKs).

The term “polymorph” refers to a solid phase of a substance, whichoccurs in several distinct forms due to different arrangements and/orconfirmations of the molecules in crystal lattice. Polymorphs typicallyhave different chemical and physical properties.

The term “pharmaceutically acceptable excipient” refers to any substanceother than a compound of the invention, added to a pharmaceuticalcomposition.

The term “pharmaceutical composition” refers to a mixture of one or moreof the salts of the present invention or the polymorphs of such salts,as described herein, with other chemical components, such asphysiologically/pharmaceutically acceptable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

The term “physiologically/pharmaceutically acceptable carrier” refers toa carrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

The term “polymorph” may also be defined as different unsolvated crystalforms of a compound. The term also includes solvates (i.e., formscontaining solvent, or water), amorphous forms (i.e., noncrystallineforms), and desolvated solvates (i.e., forms which can only be made byremoving the solvent from a solvate).

The term ‘solvate’ is used to describe a molecular complex comprising acompound of the invention and one or more pharmaceutically acceptablesolvent molecules, for example, ethanol. The term ‘hydrate’ is employedwhen said solvent is water.

The term “substantially free” in relation to the amount of a certainpolymorph in a sample means that other polymorphs are present in anamount less than about 15 weight percent. In another embodiment,“substantially free” means less than about 10 weight percent. In anotherembodiment, “substantially free” means less than about 5 weight percent.In still another embodiment, “substantially free” means less than about1 weight percent. Someone with ordinary skill in the art wouldunderstand that the phrase “in an amount less than about 15 weightpercent” means that the polymorph of interest is present in an amount ofmore than about 85 weight percent. Likewise, the phrase “less than about10 weight percent” means that the polymorph of interest is present in anamount of more than about 90 weight percent, and so on.

The term “therapeutically effective amount” refers to that amount of thecompound being administered which will prevent, alleviate, or ameliorateone or more of the symptoms of the disorder being treated, or prolongthe survival of the subject being treated. In reference to the treatmentof cancer, a therapeutically effective amount refers to that amountwhich has the effect of:

-   -   (1) reducing the size of the tumor;    -   (2) inhibiting (that is, slowing to some extent, or stopping)        tumor metastasis;    -   (3) inhibiting (that is, slowing to some extent, or stopping)        tumor growth, and/or,    -   (4) relieving to some extent (or eliminating) one or more        symptoms associated with the cancer.

Different salt forms of Compound I may be synthesized to obtain a formwith better physical properties. The base compound may be in solution.The solution is generally a solvent. In one embodiment, the solution isan alcohol. In another embodiment, the solvent may be isopropanol,methanol, acetonitrile, or water plus acetonitrile. The solution mayalso comprise a mixture of solvents.

The salts may be crystallized using a stoichiometricaddition/crystallization technique. A stoichiometric amount of thecounterion is introduced to the base in solution. In one embodiment, theamount of counterion is in a 1:1 ratio to the base. In anotherembodiment, the amount of counterion is from 0% to about 60% molarexcess to the base. In another embodiment, the amount of counterion isfrom about 10% to about 50% molar excess to the base. In yet anotherembodiment, the amount of counterion is about 40% molar excess to thebase. The counterions may include hydrochloride, fumarate, citrate,phosphate, and ascorbate ions. In one embodiment, the counterion is thephosphate ion. In another embodiment, the counterion is the citrate ion.

The salt in solution is then forced to crystallize by a variety ofcommon techniques including cooling, evaporation, drowning, etc., knownto one skilled in the art. Excess solvents may be removed from thesamples by methods known to one skilled in the art. In one embodiment,the solvents are removed from the solution by adding acetonitrile (ACN)and rotovapping the solution. The solution may be rotovapped from about40° C. to about 60° C. In another embodiment, additional solvents may beadded to the solution (eg, isopropanol and methyl ethyl ketone) prior torotovapping. The crystallizations may be conducted in the dark toprevent light-induced isomerization. In one embodiment, the crystals areremoved by filtration. In another embodiment, filtration may beperformed at ambient laboratory atmosphere.

By these methods, the ascorbate, citrate, fumarate, hydrochloride, andphosphate salts of Compound I were crystallized. Specific examples ofcrystallization methods are provided below. HPLC analysis may be used todetermine purity of the resultant sample. The physical properties of thecompounds may be determined by tests known to one skilled in the art,including melting point determination, powder X-ray diffraction, anddynamic moisture sorption gravimetry. Parameters for these tests aredescribed below.

These five salt forms are described herein (See Table 1). These salts ofCompound I are often hygroscopic. For example, as can be seen in Table1, at 80 percent humidity, the hydrochloride salt was about 20 percentwater, the fumarate salt was about 9 percent water, and the ascorbatesalt was about 6.5 percent water. This characteristic can make use ofthe salt in a pharmaceutical formulation difficult and can shorten theshelf-life of a formulation. However, two salts, the phosphate andcitrate salts, were unexpectedly found to have low moisture uptake,having about 1 percent and about 3.8 percent water at 80 percentrelative humidity, respectively.

Based on characterization of these salts, the 1:1 phosphate salt,Compound I phosphate, was identified as a salt form with highlydesirable characteristics, including good crystallinity, low moistureuptake, ease of crystallization, good purity, and lack of hydrate. Tenpolymorphs of Compound I phosphate, herein named Forms I through X, arealso described. The citrate salt also demonstrated desirablecharacteristics, such as low moisture uptake and good crystallinity.

Polymorphs of the compounds of the present invention are desirablebecause a particular polymorph of a compound may have better physicaland chemical properties than other polymorphic forms of the samecompound. For example, one polymorph may have increased solubility incertain solvents. Such added solubility may facilitate formulation oradministration of the compounds of the present invention. Differentpolymorphs may also have different mechanical properties (e.g.,different compressibility, compactibility, tabletability), which mayinfluence tableting performance of the drug, and thus influenceformulation of the drug. A particular polymorph may also exhibitdifferent dissolution rate in the same solvent, relative to anotherpolymorph. Different polymorphs may also have different physical(solid-state conversion from metastable polymorph to a more stablepolymorph) and chemical (reactivity) stability. An embodiment of thepresent invention contemplates the Form I polymorph of Compound Iphosphate, as described herein.

In embodiments of the present invention, pure, single polymorphs as wellas mixtures comprising two or more different polymorphs arecontemplated. A pure, single polymorph may be substantially free fromother polymorphs.

Some embodiments of the present invention contemplate pharmaceuticalcompositions comprising one or more of the salts of Compound I or thepolymorphs of such salts, as described herein, and a pharmaceuticallyacceptable carrier or excipient.

Polymorphs were generated from concentrated solutions of Compound Iphosphate. The concentrated solutions may be in a range of 60 to 100 mgof Compound I phosphate per mL of solution. In one embodiment, about 70mg of Compound I may be dissolved in 1 mL of phosphoric acid.

The polymorph crystals may be precipitated from a solvent by variousmethods including, for example, slow evaporation, cooling asupersaturated solution, precipitation from anti-solvents, etc., whichare known to one skilled in the art. In one embodiment, the polymorphcrystals are generated by adding the solution to an anti-solvent. Theanti-solvent may be water plus acetonitrile (ANC), ethanol, methanol,acetone, acetonitrile, THF, ethyl acetate, hexane, methylene chloride(CH₂C1₂), isopropyl alcohol (IPA), methyl ethyl ketone (MEK), anddioxane. In one embodiment, an additional solvent (eg, methanol) may beadded. In another embodiment, the samples are allowed to stand overnightprior to removing the crystals. In yet another embodiment, the samplesare allowed to stand for three days prior to removing the crystals.

The crystals may be characterized using standard methods known to oneskilled in the art, including PXRD dynamic moisture sorption gravimetry,differential scanning calorimetry, thermal gravimetric analysis, andoptical microscopy. These techniques are described below.

Pharmaceutical compositions suitable for the delivery of compounds ofthe present invention and methods for their preparation will be readilyapparent to those skilled in the art. Such compositions and methods fortheir preparation may be found, for example, in Remington'sPharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

The choice of a pharmaceutically acceptable excipient will to a largeextent depend on factors such as the particular mode of administration,the effect of the excipient on solubility and stability, and the natureof the dosage form. Examples of excipients include, without limitation,calcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

Carriers and excipients for formulation of pharmaceutically acceptablecompositions comprising Compound I are well known in the art and aredisclosed, for example, in U.S. Pat. No. 6,573,293, which isincorporated herein in its entirety. Methods of administration for suchare also known in the art and also described, for example, in U.S. Pat.No. 6,573,293. Similar methods could also be used to formulate andadminister pharmaceutically acceptable compositions of the salts ofCompound I, or the polymorphs of such salts, of this invention.

Proper formulation is dependent upon the route of administration chosen.For injection, the compounds of the present invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks' solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For parenteral administration, e.g., bybolus injection or continuous infusion, formulations may be presented inunit dosage forms, such as in ampoules or in multi-dose containers. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulating materials suchas suspending, stabilizing, or dispersing agents.

The compounds of the invention may be administered directly into theblood stream, into muscle, or into an internal organ. Suitable means forparenteral administration include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular, intrasynovial andsubcutaneous. Suitable devices for parenteral administration includeneedle (including microneedle) injectors, needle-free injectors andinfusion techniques. Parenteral formulations are typically aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably adjusted to a pH of from 3 to 9), but, forsome applications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water. Additionally,suspensions of the compounds of the present invention may be prepared ina lipophilic vehicle. Suitable lipophilic vehicles include fatty oilssuch as sesame oil, synthetic fatty acid esters, such as ethyl oleateand triglycerides, or materials such as liposomes.

The compounds of the invention may be administered orally. Oraladministration may involve swallowing, so that the compound enters thegastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth. For oral administration, the compounds can be formulatedby combining the compounds of the present invention withpharmaceutically acceptable carriers well known in the art. Formulationssuitable for oral administration include solid, semi-solid and liquidsystems such as tablets; soft or hard capsules containing multi- ornano-particulates, liquids, or powders; lozenges (includingliquid-filled); chews; gels; fast dispersing dosage forms; films;ovules; sprays; and buccal/mucoadhesive patches.

The compounds of the invention may also be administered topically,(intra)dermally, or transdermally to the skin or mucosa. Typicalformulations for this purpose include gels, hydrogels, lotions,solutions, creams, ointments, dusting powders, dressings, foams, films,skin patches, wafers, implants, sponges, fibres, bandages andmicroemulsions. Liposomes may also be used. Typical carriers includealcohol, water, mineral oil, liquid petrolatum, white petrolatum,glycerin, polyethylene glycol and propylene glycol. Penetrationenhancers may be incorporated—see, for example, J Pharm Sci, 88 (10),955-958, by Finnin and Morgan (October 1999). Other means of topicaladministration include delivery by electroporation, iontophoresis,phonophoresis, sonophoresis and microneedle or needle-free (e.g.Powderject™, Bioject™, etc.) injection.

The compounds of the present invention may be formulated for rectaladministration, such as suppositories or retention enemas using, forexample, conventional suppository bases such as cocoa butter or otherglycerides.

The compounds of the present invention may also exist in unsolvated andsolvated forms.

The embodiments of the present invention also contemplate a method forthe modulation of the catalytic activity of a PK comprising contactingsaid PK with a one or more of the salts of Compound I or the polymorphsof such salts of the present invention. Such “contacting” can beaccomplished “in vitro,” i.e., in a test tube, a petri dish, or thelike. In a test tube, contacting may involve only a compound and a PK ofinterest or it may involve whole cells. Cells may also be maintained orgrown in cell culture dishes and contacted with a compound in thatenvironment. In this context, the ability of a particular compound toaffect a PK-related disorder, i.e., the IC₅₀ of the compound, definedbelow, can be determined before use of the compounds is attempted invivo with more complex living organisms. For cells outside the organism,multiple methods exist, and are well-known to those skilled in the art,to get the PKs in contact with the compounds including, but not limitedto, direct cell microinjection and numerous transmembrane carriertechniques.

Embodiments of the present invention contemplate a method for treatingor preventing a protein kinase related disorder in an organism (e.g., acompanion animal or a human) comprising administering a therapeuticallyeffective amount of a pharmaceutical composition comprising one or moreof the salts of Compound I or the polymorphs of such salts of thepresent invention and a pharmaceutically acceptable carrier or excipientto the organism.

In an embodiment of the present invention, the protein kinase relateddisorder is selected from the group consisting of a receptor tyrosinekinase related disorder, a non-receptor tyrosine kinase relateddisorder, and a serine-threonine kinase related disorder. In anotherembodiment of the present invention, the protein kinase related disorderis selected from the group consisting of an EGFR related disorder, aPDGFR related disorder, an IGFR related disorder, and a FLK relateddisorder.

The receptor protein kinase whose catalytic activity is modulated by acompound of this invention is selected from the group consisting of EGF,HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRct, PDGFR13, CSFIR, C-Kit,C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R andFGFR-4R. The cellular tyrosine kinase whose catalytic activity ismodulated by a compound of this invention is selected from the groupconsisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak, Jak, Ack,Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. The serine-threonine proteinkinase whose catalytic activity is modulated by a compound of thisinvention is selected from the group consisting of CDK2 and Raf.

In yet another embodiment of the present invention, the protein kinaserelated disorder is selected from the group consisting of squamous cellcarcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer,bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostatecancer, breast cancer, small cell lung cancer, glioma, colorectalcancer, genitourinary cancer, gastrointestinal cancer, mastocytosis, andmast cell tumors. In an embodiment of the present invention, the proteinkinase related disorder is selected from the group consisting ofdiabetes, an autoimmune disorder, a hyperproliferation disorder,restenosis, fibrosis, psoriasis, von Heppel-Lindau disease,osteoarthritis, rheumatoid arthritis, angiogenesis, an inflammatorydisorder, an immunological disorder, and a cardiovascular disorder.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, e.g., the modulationof PK activity or the treatment or prevention of a PK-related disorder.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any compound used in themethods of the invention, the therapeutically effective amount or dosecan be estimated initially from cell culture assays. Then, the dosagecan be formulated for use in animal models so as to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture. Such information can then be used to more accuratelydetermine useful doses in humans or companion animals.

In practice, the amount of the compound to be administered ranges fromabout 0.001 to about 100 mg per kg of body weight, such total dose beinggiven at one time or in divided doses. The amount of a compositionadministered will, of course, be dependent on the subject being treated,the severity of the affliction, the manner of administration, thejudgment of the prescribing physician or veterinarian, etc. In cases oflocal administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration andother procedures known in the art may be employed to determine thecorrect dosage amount and interval.

Embodiments of the present invention also contemplate a method oftreating cancer in companion animals comprising administering apharmaceutical composition comprising one or more of the salts ofCompound I or the polymorphs of such salts of the present invention anda pharmaceutically acceptable carrier or excipient.

Additionally, it is contemplated that the salts of Compound I or thepolymorphs of such salts, as described herein, would be metabolized byenzymes in the body of an organism such as a companion animal or a humanbeing to generate a metabolite that can modulate the activity of theprotein kinases. Such metabolites are within the scope of the presentinvention.

Compounds of the invention may be administered alone or in combinationwith one or more other compounds of the invention or in combination withone or more other drugs (or as any combination thereof). It is alsocontemplated that the salts of Compound I or the polymorphs of suchsalts, as described herein, might be combined with otherchemotherapeutic agents for treatment of the diseases and disordersdiscussed above. For example, a compound of the present invention may becombined with fluorouracil alone or in further combination withleukovorin or other alkylating agents. A compound of the presentinvention may be used in combination with other antimetabolitechemotherapeutic agents such as, without limitation, folic acid analogsor the purine analogs. A compound may also be used in combination withnatural product based chemotherapeutic agents, antibioticchemotherapeutic agents, enzymatic chemotherapeutic agents, platinumcoordination complexes, and hormone and hormone antagonist. It is alsocontemplated that a compound of the present invention could be used incombination with mitoxantrone or paclitaxel for the treatment of solidtumor cancers or leukemias.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, practice the present invention toits fullest extent. The following detailed examples describe how toprepare the various compounds and/or perform the various processes ofthe invention and are to be construed as merely illustrative, and notlimitations of the preceding disclosure in any way whatsoever. Thoseskilled in the art will promptly recognize appropriate variations fromthe procedures both as to reactants and as to reaction conditions andtechniques.

EXAMPLES Example 1 Synthesis of Compound I, ie.5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-vlidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide

As described in U.S. Pat. No. 6,574,293 (example 129)5-Fluoro-1,3-dihydro-indol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-pyrrolidin-1-yl-ethyl)-amide to give Compound I.

Scale-Up Procedure. 5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(61 g), 5-fluoro-1,3-dihydro-indo1-2-one (79 g), ethanol (300 mL) andpyrrolidine (32 mL) were refluxed for 4.5 hours. Acetic acid (24 mL) wasadded to the mixture and refluxing was continued for 30 minutes. Themixture was cooled to room temperature and the solids collected byvacuum filtration and washed twice with ethanol. The solids were stirredfor 130 minutes in 40% acetone in water (400 mL) containing 12 Nhydrochloric acid (6.5mL). The solids were collected by vacuumfiltration and washed twice with 40% acetone in water. The solids weredried under vacuum to give5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (86 g, 79% yield) as an orangesolid.

5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (100 g) and dimethylformamide(500mL) were stirred andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(221 g), 1-(2-aminoethyl)pyrrolidine (45.6 g) and triethylamine (93 mL)were added. The mixture was stirred for 2 hours at ambient temperature.The solid product was collected by vacuum filtration and washed withethanol. The solids were slurry-washed by stirring in ethanol (500 mL)for one hour at 64° C. and cooled to room temperature. The solids werecollected by vacuum filtration, washed with ethanol, and dried undervacuum to give 5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-pyrrolidin-1-yl-ethyl)-amide (101.5 g, 77% yield).

Example 2 Synthesis of Salts of Compound I Example 2A Compound IPhosphate

2.67 mMoles of Compound I was added to a flask with 40 mL 0.092 Mphosphoric acid (about a 40% molar excess assuming a 1:1 salt) and 40 mLisopropanol. Then, acetonitrile was continuously added to the aqueoussolution in 30 mL aliquots, as the solution was rotovapped at 60° C. toremove the water. In all, 120 mL of acetonitrile was used to remove thewater from the solution. The crystals were filtered and air-dried.Crystals were free flowing and orange; 1.09 grams were collected for an83% yield.

Example 2B Compound I Citrate

2.64 mMoles of Compound I was added to a flask with 34 mL 0.1M citricacid (3.4 mMoles) and 35 mL methanol. This solution was rotovapped at50° C. Reducing the volume of this solution produced crystals of poorcrystallinity, so 20 mL isopropanol and 10 mL of methyl ethyl ketonewere added to dissolve the solid. This mixture was rotovapped at 60° C.and produced orange crystals. The crystals were filtered and air-dried.The yield for this process was about 60%, and could have been improvedby reducing the solvent volume further before filtration.

Example 3 Physical Properties of Salts of Compound I

Methods. Tests to determine the physical properties of the salts ofCompound I included melting point determination, HPLC purity, powderX-ray diffraction, and dynamic moisture sorption gravimetry.

Powder X-ray Diffraction (PXRD).

Powder XRD was performed using a Scintag X2 Advanced Diffraction System(lab 259-1088, controlled by Scintag DMS/NT 1.30a and Microsoft WindowsNT 4.0 software. The system uses a Copper X-ray source (45 kV and 40 mA)to provide CuKα₁ emission of 1.5406A and a solid-state Peltier cooleddetector. The beam aperture was controlled using tube divergence andanti-scatter slits of 2 and 4 mm and detector anti-scatter and receivingslits of 0.5 and 0.2 mm width. Data were collected from 2 to 35°two-theta using a step scan of 0.03°/step with a counting time of onesecond per step. Scintag round, top loading stainless steel sampleholders with 9 mm diameter inserts were utilized for the experiments.Powders were packed into the holder and were gently pressed by a glassslide to ensure coplanarity between the sample surface and the surfaceof holder.

Dynamic Moisture Sorption Gravimetry (DMSG).

DMSG isotherm was collected on a temperature controlled atmosphericmicrobalance. Approximately 10 mg samples were placed in the sample panof the balance. The humidity was sequentially varied from room relativehumidity (RH) to 0% RH and was then increased to 90% RH followed by adecrease of RH to 0% again in 3% RH steps. The mass was then measuredevery two minutes. The RH was stepped to the next target value whenchange of the sample mass was less than 0.5 μg in 10 min. The VisualBasic program dmsgscn2.exe was used to control the data collection andexport the information to an Excel spreadsheet.

Results. Table 1 shows a summary of data for the ascorbate, citrate,fumarate, hydrochloride, and phosphate salts of Compound I. HPLCanalysis suggested that the salts were of relatively high purity, and nosignificant change in the purity was induced through the salt formationprocess.

TABLE 1 Summary of salts synthesized for Compound I Melting % water atSalt point 80% HPLC % Counterion Crystallized (° C.) RH purity* Isomer*none (free NA 257 ~1% 97.8 0.63 base) hydrochloride Yes  96 ~20%  97.72.29 fumarate Yes NA ~9% 97.3 1.96 citrate Yes 181 ~3.8%   98.2 1.38phosphate Yes 288 ~1% ascorbate Yes 245 ~6.5%   *area percent under thepeaks in the HPLC

The hydrochloride, fumarate, and ascorbate salts were very hygroscopic(see FIG. 1). The other two salts (citrate and phosphate) had lowermoisture sorption profiles, absorbing less than 3% water at 70% relativehumidity.

The powder X-ray patterns indicated that the phosphate and citrate saltswere of relatively high crystallinity. (see Tables 2 and 3; and FIG. 2).

TABLE 2 PXRD Peaks of Compound I Phosphate Relative Two-Theta Angle*Intensity** (degree) (arbitrary) 11.1 23.5 11.5 24.0 11.6 19.9 12.0 8.913.0 22.0 14.0 25.9 14.5 7.0 15.4 15.0 16.5 7.5 17.2 27.7 17.8 9.5 18.316.6 19.4 11.6 19.6 11.2 19.8 9.8 20.8 58.2 21.6 11.8 22.0 9.5 22.7 9.222.9 12.4 23.2 12.8 23.4 10.3 24.4 48.8 25.8 30.4 27.0 100.0 29.2 7.429.2 7.4 33.5 5.1 *±0.1° **The relative intensity for each peak isdetermined by normalizing its intensity to that of the strongest peak at27.0° angle as 100

TABLE 3 PXRD Peaks of Compound I Citrate Relative Two-Theta Angle*Intensity** (degree) (arbitrary) 3.2 20.4 7.3 17.7 9.2 14.4 9.5 39.110.8 9.5 12.7 23.5 13.2 19.6 14.3 23.5 14.8 16.7 15.9 11.3 17.2 8.3 17.811.9 18.2 19.8 18.3 19.5 19.6 6.8 20.9 11.1 21.6 8.9 21.7 10.5 21.8 8.923.2 13.2 24.2 20.3 24.9 15.9 25.5 100.0 26.4 20.9 26.8 26.5 27.1 10.832.0 6.3 34.4 8.1 *±0.1° **The relative intensity for each peak isdetermined by normalizing its intensity to that of the strongest peak at25.5° angle as 100

Example 4 Preparation and Characterization of Compound I PhosphateExample 4A Preparation of Compound I Phosphate

Compound I free base was used to prepare the phosphate salt. A sample(lot number 35282-CS-51) of Compound I phosphate was prepared asdescribed above. 4 mL of 0.977 M phosphoric acid was added to 1.095 g offree base in a flask immediately followed by adding 4 mL ofacetonitrile. A suspension was obtained. The suspension was heatedslightly on a hot-plate. Adding 40 mL of water and heating whilestirring for about one hour did not completely dissolve the solid. Thesolid was filtered and washed with 10 mL of acetonitrile. PXRD showed itwas the phosphate salt of Compound I.)

Example 4B Characterization of Compound I Phosphate

Lot 35282-CS-51 was named polymorph Form I of Compound I Phosphate. Ithas high crystallinity, good flowability, and large crystal size. Boththe absence of the melting event at the melting temperature of CompoundI free base (free base polymorph Form A, 256° C.; free base polymorphForm B, 259° C.) and the presence of high melting points (281-297° C.)of the solids suggested that the crystals of Lot 35282-CS-51 are adifferent salt form and not Compound I free base. The purity of the lotwas 99.6% by HPLC.

Example 4C Estimation of Solubility of Compound I Phosphate

Samples of 1-2 mg of Compound I phosphate (lot 35282-CS-51) weretransferred to 10 mL glass vials (tared) and were weighed (accurate to0.1 mg). Solvents were added to the vials (one solvent to each vial) ina step-wise fashion, with 0.5 mL of solvent added at each step. Solventsused were buffer (pH=2), buffer (pH=5), water, methanol, tetrahydrofuran(THF), acetonitrile, and acetone. After each addition, the vial wascapped and shaken. The dissolution of solid was visually observed. If noobvious dissolution was observed, more solvent was added immediately. Ifdissolution was apparent, the vial was left on the bench for at least 30minutes before the next addition of solvent. This step was repeateduntil no crystals were visible against a black and a white background.The solubility was then bracketed by dividing the weight of the compoundby the final volume and the volume before the last addition. If a solidremained after the addition of 10 mL of solvent, the solubility wasexpressed as less than the weight divided by the final volume. If thesolid was completely dissolved after the first addition of solvent, thesolubility was expressed as greater than the weight divided by thesolvent volume. All experiments were conducted at room temperature.

The estimated solubilities of Compound I phosphate in various solventsare presented in Table 4 along with solubilities of the free base,expressed as mg/mL. The solubility of Compound I phosphate is lower thanthat of Compound I free base in the same solvent, except in water (atvarious pH levels). The solubility of Compound I phosphate depends onthe pH value of a solution, and becomes considerably higher (>3 mg/mL)at pH 2 or lower. The melting point of Compound I phosphate (lot35282-CS-51) is about 281-297 ° C., which is substantially higher thanthe melting point of Compound I free base (free base polymorph Form A,256° C.; free base polymorph Form B, 260° C.). One important result isthat the wettability of Compound I phosphate with water is much betterthan that of Compound I free base.

TABLE 4 Estimated solubility of Compound I free base and Compound Iphosphate in various solvents at 23° C. Solubility of Compound I freebase (polymorph Solubility of Compound I Solvent Form A) (mg/mL)phosphate (mg/mL)^(d) 1 buffer (pH = 2)^(a) 3.11 5.9-7.4 2 buffer (pH =5)^(b) 0.005 3 water NA^(c) ~0.29^(e) (some particles stuck on the vialwall and did not dissolve) 4 methanol 0.21-0.31 ~0.14 5 THF 0.32-0.4 <<<0.19 6 Acetonitrile <<0.08 <<<0.13 7 Acetone <<0.16 <<<0.2 ^(a)pH = 2buffer is made of HCl and KCl ^(b)pH = 5 buffer is made of potassiumacid phthalate and sodium hydroxide. ^(c)Not available but expected<0.005 mg/mL. ^(d)1 g of Compound I Phosphate is equivalent to 0.802 gof Compound I free base. ^(e)The final solution pH value is 4.91.

Example 5 Generation of Compound I Phosphate Polymorphs

The low solubilities of Compound I phosphate seen in Example 4Cindicated that solutions of highly concentrated (60-100 mg/mL, darkorange-red) Compound I phosphate would be beneficial to precipitatepolymorphs of Compound I phosphate from various solvents. Suchconcentrated solutions were prepared by dissolving Compound I free basein about 1 M phosphoric acid. For example, about 70 mg of Compound Ifree base could be dissolved in 1 mL of 1 M phosphoric acid. However,the amount of Compound I free base and phosphoric acid used depended onthe desired concentration and batch size of the solution. In the examplein which the precipitate was vacuum filtered immediately afterprecipitation, about 1 mL of the desired solution was then dripped intoabout 10 mL of ten anti-solvents to precipitate the salt crystals out.These solvents were water plus acetonitrile (ANC), ethanol, methanol,acetone, acetonitrile, THF, ethyl acetate, hexane, methylene chloride(CH₂Cl₂), and isopropyl alcohol (IPA). In the example in which theprecipitate was vacuum filtered after standing overnight or for threedays, the additional solvents of methyl ethyl ketone (MEK) and dioxanewere used. Some organic solvents, e.g., ethyl acetate, hexane, CH₂C1₂,are not miscible with water and two layers of solvents were observed.Only a little precipitation was seen at the interface even minutes afteraddition. In those cases, about 1 mL of methanol was added as a bridgingsolvent, to increase the miscibility between the two layers. Methanolappeared to work well to increase the miscibility because colorlessorganic layer became yellow as soon as methanol was added. The vial wasthen shaken vigorously by hand for about one minute. The solidsprecipitated from organic solvents were vacuum filtered both immediatelyafter precipitation (within 20 min) and after standing overnight or forthree days in order to isolate both metastable and stable polymorphs.The powder was then analyzed. The different solids were numbered in theorder of discovery.

Example 6 Characterization of the Polymorphs of Compound I PhosphateExample 6A Characterization Methods

All powders obtained from the above polymorph screening procedures wereanalyzed by PXRD, as described in Example 3 above. When a new PXRDpattern was observed, complementary techniques were also used tocharacterize the solids, including dynamic moisture sorption gravimetry(also described in Example 3), differential scanning calorimetry,thermal gravimetric analysis (when necessary), and optical microscopy.

Differential Scanning calorimetry (DSC). DSC data were obtained using aDSC calorimeter (TA Instruments 2920). Powder (1-5 mg) was packed in analuminum DSC pan. An aluminum lid was place on top of the pan and wascrimped. The crimped pan was placed in the sample cell along with anempty pan as a reference. Temperatures were increased to 300 or 350° C.from 30° C. at a rate of 10° C/min unless otherwise specified.

Thermogravimetry (TGA). TGA experiments were performed using a highresolution analyzer (TA Instruments model 2950). The TA InstrumentsThermal Solutions™ for NT (version 1.3 L) was used for data collection,and the Universal Analysis™ for NT (version 2.4 F) was used for dataanalysis. Samples (5-10 mg) were placed onto a aluminum pan which wasfurther placed on a platinum weighing pan before being heated. Theweights of the aluminum and platinum pans were tared prior to loadingthe samples. The temperature was increased from 30° C. to 300 ° C.linearly at a rate of 10° C/min. Dry nitrogen purge was used.

Polarized Light Microscopy. Microscopy was conducted on an Olympus BHSPpolarized light microscope. Powder was suspended in silicone oil anddispersed between a microscopy slide and a cover slip. Prior toobservation, the cover slip was gently rubbed against the slide torender good dispersion of the particles.

Example 6B Characterization Immediately After Precipitation

The results are summarized in Table 5. Precipitation took place as soonas the acidic solution was mixed with the anti-solvents. At first, theprecipitates were loose flocs. The colors were yellow or light-orange ingeneral. The resulting solid was sticky. The microscopic observation ofthese solids indicated that they were constituted of very smallcrystallites with good birefringence under polarized light. At least sixdifferent PXRD patterns were observed on solids obtained from ninesolvent systems. (See FIG. 3) The amide side-chain on this molecule isflexible and it undertakes different conformations in free base Form Band in its hydrochloride salt. Therefore, the molecule in the differentsolid forms may be conformational polymorphs. The PXRD patterns ofsolids precipitated from ethyl acetate, hexane, and IPA appeared thesame. However, a detailed comparison with other PXRD patterns wasdifficult because of the low diffraction signals of solids from thesethree solvents. Consequently, they were not assigned as a new form. Theprecipitate from methanol is the same as the reference lot 35282-CS-51(assigned as Form I). TGA data of all precipitates indicated residuesolvent at a level of 1.7-4.7%. Of these solids, the one from CH₂Cl₂appeared to be a solid with retained solvent in crystals. The TGA curveshowed an abrupt decrease in sample weight at a temperature about 125°C. (see FIG. 4). This event is recorded as an endotherm at about thesame temperature by DSC. In addition, this powder was constituted ofcrystals of well-defined morphology and was free-flowing, a verydifferent property from other lots of precipitates. The powder exhibitedmedium crystallinity by PXRD but good crystallinity when observed bypolarized-light microscope. Other lots were constituted of very finecrystallites. On DSC curve of these powders, a broad and shallowendotherm was seen as soon as the sample was loaded to the sample cell.This observation is reflected by TGA as a gradual weight loss from thebeginning of heating on TGA. Therefore, for these lots, the residualsolvents were probably surface adsorbed solvents and were not solventsin the crystal lattice.

TABLE 5 Physical characterization of solids isolated immediately afterprecipitation Free Name TGA flowing? Assigned to Organic PXRDMicroscopic wt % Thermal events Color of (Y = yes; PXRD pattern solventcrystallinity observation loss^(a) by DSC (° C.)^(b) the solid N = no)Form I Water + ACN High Irregular 0.3 296 Orange Y Form II EtOH PoorAggregate^(e) 2.5 81^(c), 226, 293 Yellow N (sticky) Form I MeOH HighIrregular 1.7 298 Orange-red Y Form III Acetone Medium Aggregate^(e) 2.588^(c), 155, 222, Yellow N (sticky) 228, 286 Form IV Acetonitrile HighAggregate^(e) 2.9 97^(c), 142, 156, Yellow N (sticky) (ACN) 172, 227,230, 281 Form V THF High Aggregate^(e) 2.5 82^(c), 161, 175, Yellow N(sticky) 227, 292 ND^(f) Ethyl acetate Poor Fine 4.7 98^(c), 196, 222Light- N (sticky) particles orange ND^(f) Hexane Poor Aggregate^(e)Light- N (sticky) Orange Form VI^(g) CH₂Cl₂ Medium Plate + 3.3 123^(d),164, 228, Orange-red Y equant + 296 fine ND^(f) IPA Poor Aggregate^(e)1.9 76^(c), 222, 229, yellow N (sticky) 296 ^(a)Weight loss up to 160°C. ^(b)For clarity, the temperatures listed in the table are peaktemperatures only. ^(c)Peak temperatures of a broad endothermal eventfrom the starting temperature of a DSC run. These events are alsoreflected by gradual weight loss on TGA. This type of heat event istypical of loss of surface adsorbed solvent. ^(d)Peak temperatures of arelatively sharp endothermal event on DSC. This event is also recordedby TGA as a sudden loss of weight at the corresponding peak temperature.This type of heat event is typical of desolvation of a solvate. ^(e)Theaggregates are constituted of very fine particles. ^(f)ND means that nodefinite form can be assigned because of the low signal of thediffraction peaks of corresponding PXRD patterns obscured a detailedcomparison with PXRD patterns of other forms. ^(g)Form VI, is a solventcontaining solid.

Example 6C Precipitation After Standing up to Three Days in Solvent

These results are summarized in Table 6. After a standing time of up tothree days in the solvent, a new non-fluffy orange-red solid phaseappeared. The fluffy precipitates obtained from all organic solvents,except the precipitate from methanol, underwent transformation.Apparently, the solids precipitated immediately after precipitation weremetastable in these cases in that they converted to a more stable solidform (Form I) over time. This conversion appeared to be completed in acouple of hours in most of the solvent systems. However, they wereallowed to stand for a much longer period of time to ensure thecompletion of the process in order to avoid reaping a mixture of twosolid forms. The TGA curve showed abrupt weight loss at about 124° C.and 153° C. for the solids obtained from hexane and acetonitrilerespectively, coupled by an endotherm at a similar temperature on DSC.Therefore, they also appeared to contain restrained solvent in crystallattice. The stoichiometries of the retained solvents are about 0.6 foracetonitrile and about 0.14 for hexane. Needle-shaped crystals weregrown from acetonitrile after standing for three days. The PXRD patternsof the acetonitrile-retaining solid were unique while the PXRD patternof the hexane solvate is similar to the CH₂Cl₂-retaining solididentified earlier (FIG. 3). Both solvent-retaining solids (hexane andacetonitrile) lost weight on a TGA pan. Unique PXRD patterns of bothsolids were observed after the corresponding retained solvent had beenremoved by heating (Table 7, FIG. 3), indicating that removal of solventmolecules from the solids caused structural changes of the solvatecrystals (therefore, the solvent molecules are in crystal lattice notjust on crystal surfaces). However, the PXRD pattern of acetonitriledesolvate was low in signal intensity. DSC profile of the acetonitriledesolvate exhibited two additional heat events at 74° C. and 174° C.,when compared with the DSC profile of the acetonitrile solvate, whilethe desolvation event at 153° C. was absent. Cooling of the sample afterdesolvation may have changed the solid that undergoes an energeticchange at 174° C.

When other organic solvents were used, the longer standing period of theprecipitates yielded solids of the same PXRD pattern as that of Form I(Lot 35282-CS-51) although the morphology of the crystals was different(Table 6). The same PXRD pattern indicated that those solids have thesame crystal lattice structure. The different morphology must be due tothe solvent effects. It is apparent that Form I is the most stable solidphase among all non-solvated polymorphs reported herein. Othersolvent-free solid forms were metastable and converted to Form I quicklywhen in contact with solvent. The solid from CH₂Cl₂ appeared to flowmore easily than the solid from hexane. The TGA, morphology, and theflowability indicated that they are two different solids.

TABLE 6 Physical characterization of solids isolated after varyingstanding periods after precipitation Name Assigned Organic solventThermal to PXRD (standing PXRD Microscopic wt % events by Color of Freeflowing? Pattern period) crystallinity observation loss^(a) DSC (°C.)^(b) the solid (Y = yes; N = no) Form I Water + ACN High irregular0.3 296 Orange Y Form I Ethyl acetate High Tablets Orange-red Y(overnight) Form VII^(g) Hexane Medium plates 2.4 124^(c), 228,Orange-red N (overnight) 295 Form I IPA High equant (25 0.4 297Orange-red Y (overnight) um) + fine Form I CH₂Cl₂ High equant (25 0.6293 Orange-red Y (overnight) um) + fine Form I EtOH High Aggregate^(e)Orange-red^(d) Y (3 days) Form I Acetone High plates Orange-red^(d) Y (3days) Form VIII^(g) Acetonitrile Medium needles 4.9 153^(c), 229,Orange-red^(e) N (3 days) 231, 239, 291 Form I THF High Spear head 0.3295 Light N (3 days) orange^(f) Form I MeOH high Orange-red Y(overnight) Form I MEK high rods 0.6 297 orange N (overnight) Form Idioxane high plates 295 orange N (overnight) ^(a)Weight loss up to 160°C. by TGA. ^(b)For clarity, the temperatures listed in the table arepeak temperatures only. ^(c)Peak temperatures of a relatively sharpendothermal event on DSC. This event is also recorded by TGA as a suddenloss of weight at the corresponding peak temperature. ^(d)Conversion wascomplete in 5 hrs (from yellow to orange-red). ^(e)Conversion was notobvious in 5 hrs but was mostly completed in three days (yellow looseprecipitate to orange-red well-formed needle-shaped crystals).^(f)Conversion was complete in 5 hrs. The color change was not obvious(from yellow to light orange). ^(g)Form VII and VIII are solventcontaining solids.

TABLE 7 Physical characterization of solids produced after desolvation.Name Method of Thermal Color Assigned to crystal PXRD events by of thePXRD pattern generation crystallinity DSC (° C.) solid Form IXDesolvation of Poor 74, 174, 229, orange ACN solvate 231, 239, 291 FormX Desolvation of medium orange hexane solvate

Example 7 Inhibition of KIT Phosphorylation in Canine Mast Cell Tumors

Purpose. The development of targeted therapies for cancer offers theopportunity to directly evaluate drug effects on the molecular targetand correlate these effects with tumor biology and drugpharmacokinetics. This can be instrumental in oncology drug developmentbecause it establishes a pharmacodynamic/pharmacokinetic relationshipand provides critical information regarding the therapeutic impact of atargeted agent. The purpose of this study was to evaluate the effect ofa single dose of the receptor tyrosine kinase inhibitor Compound Iphosphate on the activity of its molecular target KIT in canine mastcell tumors (MCT), in canine patients with advanced MCTs using KITphosphorylation as a marker of direct target inhibition. Also studiedwas phosphorylation of ERK1/2 (a mitogenactivated protein kinase (MAPK)downstream of KIT signaling), Compound I phosphate plasma concentration,and the mutational status of c-kit to determine how these parameterscorrelate with KIT phosphorylation status after Compound I phosphatetreatment.

Study Drug. Compound I phosphate was available in 20-mg scored tablets.

Study Design. This study was a proof of target modulation study in dogswith recurrent or metastatic grade MCTs. Patients received a single oraldose of Compound I phosphate at 3.25 mg/kg. Using a 6-mm punch biopsyinstrument, samples were obtained from the tumor before Compound Iphosphate administration and 8 hours (h) after treatment. When possible,multiple biopsies were taken. Each sample was flash frozen in liquidnitrogen and stored at −70° centigrade (C) before analysis. Bloodsamples for analysis of plasma levels of Compound I phosphate wereobtained at the same time as tumor biopsies (see below).

Compound I Phosphate Plasma Levels. Blood samples were drawn from thejugular vein and placed into a red-top serum collection vacuum glasstube. Specimens were kept at room temperature, allowed to clot,centrifuged at 1500 rpm at 4° C. for 10 minutes, transferred tocryovials, and plasma frozen at −70° C. pending analysis. Briefly,plasma samples (20 μl) or Compound I phosphate standards in canineplasma were mixed with methanol (200 μl) containing DL-propranololhydrochloride (internal standard) in a 96-well polypropylene plate(Orochem Technology, Westmont, Ill.). The plate was mixed by vortex for1 min, and the samples were centrifuged for 10 min at 4000 rpm. Tenmicroliters of the supernatant were injected onto the LC/MS/MS system,in which separation occurred on a BataBasic C-18 (5 μm, 100×4.6 mm)reverse-phase high-performance liquid chromatography column (KeystoneScientific, Foster City, Calif.). The amount of Compound I phosphate andthe internal standard in each canine plasma sample were quantified basedon standard curves generated using known amounts of compound rangingfrom 0.2 to 500 ng/ml.

c-kit Mutation Analysis. For the majority of the samples, RNA wasextracted using TRIzol (Invitrogen, Carlsbad, Calif.) according to themanufacturer's specifications. cDNA was then generated from the RNAusing dNTPs, random primers, 5× First Strand Buffer, 0.1 M DTT, andSuperscript Taq polymerase (all from Promega, Madison, Wis.). The cDNAwas quantified for each sample. For the remaining samples, genomic DNAwas prepared as described previously (Downing, S., Chien, M. B., Kass,P. H., Moore, P. F., and London, C. A. Prevalence and importance ofinternal tandem duplications in exons 11 and 12 of c-kit in mast celltumors of dogs. Am. J. Vet. Res., 63: 1718-1723, 2002; which isincorporated by reference in its entirety). For both reactions, the PCRwas run for 40 cycles consisting of 94° C. (1 min), 59° C. (1 min), and72° C. (1 min), with a 5 min 72° C. extension at the end of thereaction. A c-kit cDNA generated from the canine C2 mast cell line andcDNA generated from normal canine cerebellum were used as controls.

The PCR products were separated by electrophoresis on a 4% agarose gel;the expected wild-type c-kit PCR product is 196 by in size for PCR fromcDNA and 190 by in size for genomic DNA PCR. For those cases in which anITD was not obvious (only a single band was present), the PCR productswere gel purified using the Promega PCR Wizard Clean-Up kit (Promega)and sequenced using both P1 (forward) and P5 or P2 (reverse) primers atthe core sequencing facility at the University of California—Davis, torule out the presence of very small ITDs, deletions, or point mutations.Sequence alignment and comparison were performed using the DNASISsequence analysis program.

Analysis of KIT and ERK Phosphorylation. Tumor biopsies were frozen inliquid nitrogen and later pulverized using a liquid nitrogen-cooledcryomortar and pestle, then stored at −70° C. until used. For theanalysis of KIT, pulverized tumors were homogenized, lysed, andimmunoprecipitated from 1 mg of starting tumor lysate, as describedpreviously (Abrams, T. J., Lee, L. B., Murray, L. J., Pryer, N. K.,Cherrington, J. M. SU11248 inhibits KIT and platelet-derived growthfactor receptor beta in preclinical models of human small cell lungcancer. Mol. Cancer Ther. 2: 471-478, 2003; which is incorporated byreference in its entirety) using an agarose-conjugated antibody to KIT(SC-1493AC; Santa Cruz Biotechnology, Santa Cruz, Calif.). When multiplebiopsies were available, repeat immunoprecipitation/Western blotanalysis was performed on separate biopsies. The amount ofphosphorylated KIT in each sample was determined by Western blot usingan antibody to phosphotyrosine 719 of murine KIT (3391; Cell SignalingTechnology, Beverly, Mass.), which corresponds to tyrosine 721 of canineKIT and is an autophosphorylation site and, thus, a surrogate for KITkinase activity. For the analysis of total KIT, the blots were stripped,reblocked, and reprobed with an antibody to KIT (A-4542; DAKO Corp.,Carpinteria, Calif.). For analysis of p42/44 ERK, the same tumor lysatesused for KIT analysis were probed by Western blot with an antibody tophospho-Thr 202/Tyr 204 ERK1/2 (9101B; Cell Signaling Technology) andthen stripped and reprobed with an antibody to total ERK (9102; CellSignaling Technology). Evaluable tumor biopsy pairs for both KIT andERK1/2 were considered those for which detectable total protein waspresent in both biopsies of the pair. Target modulation was scored byeye by three observers blinded to the JM status and plasmaconcentration. Reduction of ≧50% in phospho-protein signal relative tototal protein signal in the biopsy sample taken post-treatment comparedwith the pretreatment biopsy was scored as positive for targetmodulation, whereas a reduction of <50% was scored negative.

Results. Fourteen dogs were enrolled in this clinical study with theprimary objective to determine whether a reduction in KIT tyrosinephosphorylation occurred after oral administration of a single dose ofCompound I phosphate. KIT tyrosine phosphorylation was assessed using aphospho-specific antibody directed against an autophosphorylation sitein KIT, serving as a surrogate for KIT kinase activity. In addition,c-kit JM mutational status (ITD+ or ITD−) was determined from thebaseline tumor biopsy, and plasma concentrations of Compound I phosphatewere measured 8 hours after dosing to correlate these parameters withinhibition of KIT phosphorylation. Eleven of the 14 dogs were evaluablefor KIT target modulation. The three dogs deemed not evaluable hadundetectable or greatly reduced total KIT protein in one or bothbiopsies and so could not be scored for target modulation. The data forall dogs enrolled in the study are summarized in Table 8.

TABLE 8 Summary data for all patients enrolled Plasma c-kit ITD CompoundP-KIT P-ERK½ Patient Tumor mutation I phosphate reduction reductionnumber grade present postdose (ng/mL) postdose^(a) postdose 1 III Yes81.0 Yes No 2 III Yes 33.2 Yes No 3 II Yes 83.5 NE Yes 4 III Yes 98.0Yes Yes 5 III Yes 116.0 Yes Yes 6 III No 121.0 Yes Yes 7 III No 186.0 NENE 8 III No 0.3 Yes No 9 II No 103.0 NE NE 10 II No 111.0 No Yes 11 IINo 158.0 No Yes 12 II No 65.3 Yes Yes 13 II No 95.4 No No 14 III No119.0 Yes NE ^(a)NE, nonevaluable, P-KIT, Phospho-Tyr721 KIT, P-ERK½,Phospho-Thr202/Tyr204 ERK½

Of the 14 dogs analyzed, 5 (36%) had an ITD by PCR analysis (FIG. 5,Lanes 1-5); all five tumors had evidence of an ITD. Interestingly,patient 2 had apparently lost the wild-type c-kit allele. The PCRproducts from the remaining nine dogs that did not have evidence of anITD (FIG. 5, Lanes 6-14) were directly sequenced, and none demonstratedany type of mutation (insertion, deletion, or point mutation). For Lanes3, 6, 8, and 9, genomic DNA was used for the PCR reaction, resulting ina slightly smaller (190 bp) wild-type product.

The level of total and phosphorylated KIT expressed in the MCTs atbaseline varied between animals. Higher KIT expression correlated withhigher tumor grade. Four of eight grade III tumors had high KITexpression, compared to one of six grade II tumors (FIG. 6). Forexample, the total KIT expression in the tumor from patient 2 (gradeIII) was markedly higher than that in the tumor from patient 11 (gradeII). Dogs with grade III tumors also had a higher incidence of highlevels of phosphorylated KIT at baseline than those with grade IItumors, consistent with the increased frequency of c-kit ITD mutationsin advanced tumors and consequently elevated levels ofligand-independent phosphorylated KIT. Five of the evaluable seven dogswith grade III tumors had high levels of phosphorylated KIT at baseline;four of these were positive for the presence of an ITD in c-kit. Only 1grade II tumor had significant phosphorylated KIT; this animal alsoexpressed ITD-mutated c-kit.

Eight of the 11 evaluable dogs scored positive for target modulationusing the criterion of a ≧50% reduction in phosphorylated KIT relativeto total KIT in the biopsy sample taken after Compound I phosphatetreatment when compared with the pretreatment sample. Examples ofphosphorylated KIT and total KIT in immunoprecipitates of tumor biopsiestaken before and after treatment with Compound I phosphate are shown inFIG. 6. Five tumors (FIG. 6, left) were scored as positive for targetmodulation, whereas two tumors (FIG. 6, right) were scored as negative.Biopsy pairs that were scored as negative for inhibition of KITphosphorylation after Compound I phosphate treatment all had markedlyless phosphorylated KIT at baseline than those that scored positive(FIG. 6).

To evaluate effects of Compound I phosphate inhibition on downstreamsignaling pathways regulated by KIT phosphorylation, levels of thephosphorylated MAPK ERK1/2 were evaluated by Western blot analysis ofthe same biopsy pairs used for KIT analysis. Eleven of 14 tumors wereevaluable for phospho-ERK1/2 target modulation (two of these were alsononevaluable for KIT target modulation). Of the 11 evaluable, 7 showed areduction in the ratio of phospho-ERK1/2 to total ERK1/2 in tumorssampled after the administration of Compound I phosphate, compared withbaseline tumor samples (see FIG. 6). ERK target modulation was morefrequently detected in MCTs with relatively high baseline ERK expressionand phosphorylation than in those with low ERK.

Based on preclinical work in rodent models, the therapeutic range ofCompound I for target inhibition was considered to be 50-100 ng/ml for12 h of a 24-h dosing period. The plasma concentration of Compound Iphosphate at 8 h (approximately Cmax) after a single dose at 3.25 mg/kgranged from 33.2 to 186 ng/ml, with an average of 105±9 ng/mL (Table 8).In one animal, the plasma concentration of Compound I phosphate wasoutside the range of the other samples (0.3 ng/ml). Twelve of 14 dogshad plasma levels considered to be in the therapeutic range establishedin a Phase I clinical study of Compound I. (London, C. A., Hannah, A.L., Zadovoskaya, R., Chien M. B., Kollias-Baker, C., Rosenberg, M.,Downing, S., Post, G., Boucher, J., Shenoy, N., Mendel, D. B., andCherrington, J. M. Phase I dose-escalating study of SU11654, a smallmolecule receptor tyrosine kinase inhibitor, in dogs with spontaneousmalignancies. Clin. Cancer Res., 2755-2768, 2003) The average plasmaconcentration for dogs with evidence of KIT target modulation (79.2±41ng/ml) and those that did not score for KIT target modulation 137±36ng/ml) was not significantly different (P=0.08).

Discussion. This correlative study was designed to investigate targetmodulation in a comparable clinical population by studying the effectsof a single clinically efficacious dose of Compound I phosphate on thephosphorylation of KIT in canine MCTs and the subsequent impact onsignaling through MAPKs. The plasma concentrations of Compound Iphosphate achieved in this study were measured near the expected Cmax,based on preclinical pharmacokinetic studies and were consistent withdrug levels measured in the Phase I clinical study investigating theefficacious dose and regimen of Compound I (Table 8).

Eight of 11 (73%) evaluable MCT biopsy pairs had detectable inhibitionof KIT activation as measured by a reduction in phosphorylated KIT aftera single oral dose of Compound I phosphate. The three patients that didnot show detectable KIT target modulation after treatment had MCTs thatexpressed low levels of KIT and phospho-KIT at baseline. The lack ofsignificant target modulation in these patients may be attributable totechnical limits in the detection method; the sensitivity of thephospho-specific antibody for phosphorylated KIT relative tononphosphorylated KIT may be insufficient in samples with low baselineKIT expression. Inhibition of KIT activity correlated more closely withbaseline KIT phosphorylation than with c-kit ITD genotype. Based oncellular assays, it would be predicted that both wild-type and ITDmutant KIT would be inhibited by Compound I phosphate in vivo, becauseCompound I in vitro blocked the phosphorylation of wild-type and ITDmutant KIT with comparable potency.

Compound I phosphate also affected a signaling pathway downstream ofKIT. Mutations in c-kit in GIST and hematopoietic malignancies have beenreported to activate different signaling pathways from each other andfrom wild-type KIT. In canine MCTs, all but one tumor sample haddetectable phosphorylated ERK1/2 at baseline. In 7 of 11 evaluable tumorbiopsy pairs, ERK1/2 was inhibited, as measured by a reduction inphosphorylated ERK1/2 after treatment. Not all of the tumors scoringpositive for ERK1/2 inhibition were also positive for inhibition of KITphosphorylation. ERK1/2 target modulation did not correlate with tumorgrade or the presence or absence of c-kit ITD mutation. As for KITtarget modulation, ERK1/2 target modulation was detected more frequentlyin tumors that expressed high levels of ERK1/2 and phosphorylated ERK1/2at baseline.

The detection of inhibition of a molecular target of Compound Iphosphate after treatment of MCTs serves as proof of target modulationfor Compound I phosphate in this setting. The clinical relevance of thisfinding is supported by the correlation between inhibition of themolecular target and plasma drug concentrations in the therapeuticrange, and the previously reported clinical objective responses toCompound I in canine patients with MCTs expressing activating mutationsin the target gene, providing proof of concept for Compound I phosphatein this population of patients. Because dogs with other malignancies(including mammary carcinoma, soft tissue sarcoma, and multiple myeloma)also experienced durable objective responses on treatment with CompoundI, KIT inhibition at this plasma concentration may be reasonablyextrapolated to successful inhibition of the other closely relatedreceptor tyrosine kinase targets of Compound I expressed by thesetumors, based on in vitro and in vivo potency of Compound I, providing amolecular rationale for objective responses in these tumors. Forexample, canine mammary tumors express VEGFR, which is inhibited byindolinone tyrosine kinase inhibitors at comparable concentrations toKIT in cellular in vitro assays. (Liao, A. T., Chien, M. B., Shenoy, N.,Mendel, D. B., McMahon, G., Cherrington, J. M., and London, C. A.Inhibition of constitutively active forms of mutant kit by multitargetedindolinone tyrosine kinase inhibitors. Blood, 100: 585-593, 2002)Compound I phosphate inhibition of both wild-type and LTD mutant c-kitin MCTs can, thus, serve as a surrogate for inhibition of the relatedRTK targets of Compound I phosphate, VEGFR, and PDGFR, which areaberrantly expressed and/or regulated by many different tumor types.Finally, molecular target inhibition, coupled with clinical objectiveresponses in canine tumors, directs the development of related compoundsin human cancer toward clinical populations expressing activated KIT,VEGFR, or PDGFR.

Example 8 Multicenter, Placebo-Controlled, Double Blind, RandomizedStudy of Oral Compound I Phosphate in the Treatment of Dogs withRecurrent Mast Cell Tumors

Purpose. The effectiveness of Compound I phosphate oral tablets for thetreatment of mast cell tumors in client-owned animals that had recurrentmeasurable disease after surgery was evaluated in a masked, negativecontrolled study. The study evaluated every-other-day dosing of CompoundI phosphate at 3.25 mg free base equivalents (FBE)/kg body weight ondisease response using modified (RECIST) criteria of response. Thepresence or absence of c-kit mutation in mast cell tumors was evaluatedas a covariate in this study. For decision-making purposes, the durationof the study was 6 weeks.

One-hundred-fifty-three (153) dogs were randomized in a ratio of 4:3into one of two treatment groups: T01 (Placebo in which n=65) and T02(Compound I phosphate in which n=88). Ten veterinary oncology practicesin the United States were selected and enrolled cases. For enrollment,dogs had to have recurrent mast cell tumor (at least one target lesionhad to have a minimum longest diameter of 20 mm)±regional lymph nodeinvolvement. A maximum of three target lesions (measurable mast celltumors) and all non-target lesions (all remaining lesions, measurable orun-measurable) were identified at baseline by two evaluators. Efficacywas based on the objective response (complete response or partialresponse) at the week 6 visit where the mean of the two evaluators sumof the longest diameter of target lesions (Mean Sum LD) was compared tothe Baseline Mean Sum LD for calculation of percent reduction orincrease. Assessment of non-target lesions was subjective. A completeresponse (CR) was defined as the disappearance of all target andnon-target lesions and the appearance of no new lesions; a partialresponse (PR) was defined as at least a 30% decrease in the Mean Sum LDof target lesions compared to the Baseline Mean Sum LD andnon-progression of non-target lesions and appearance of no new lesions.Tissue samples from the tumor and distant normal skin were collectedprior to randomization and submitted for the assessment of c-kitmutation status.

Eighty-six (86) T02 and 65 T01 animals were included in the efficacyanalysis. The data analysis indicated a statistically significantimprovement in the primary endpoint (objective response) for Compound Iphosphate (T02) compared to placebo (T01). The T02 animals had asignificantly greater objective response rate (38.3%; 33/86) compared toT01 animals (7.9%; 5/63) (p<0.001). Nearly twice as many TO1 animals(66.7%; 42/63) experienced progressive disease compared to T02 animals(33.7%; 29/86). Dogs in the T02 group that were positive for the c-kitmutation were almost twice as likely to have an objective responsecompared to those that were negative for the c-kit mutation (60%, 12/20vs. 32.8%, 21/64, respectively).

In conclusion, this study demonstrated the effectiveness of Compound Iphosphate oral tablets for the treatment of recurrent mast cell tumorsin client-owned dogs.

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently, only such limitations as appear in thefollowing claims should be placed on the invention.

What is claimed:
 1. A phosphate salt of 5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide.
 2. The salt of claim 1, wherein the salt is the phosphate salt with the structure:


3. The salt of claim 2, wherein said salt has a molecular formula of C₂₂H₂₅FN₄O₂H₃PO₄ and a melting point from about 285 to about 290° C.
 4. A polymorph (Form 1) of the salt of claim 2, wherein said polymorph has a powder X-ray diffraction spectrum comprising peaks expressed in degrees (±0.1 degree) of two theta angle of 20.8, 24.5, 25.9, and 27.0 obtained using CuKα₁ emission (wavelength=1.5406 Angstroms).
 5. A pharmaceutical composition comprising a phosphate salt or a citrate salt of 5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide and a pharmaceutically acceptable carrier or excipient. 